This is a proposal to acquire an Amnis ImageStream high speed imaging flow cytometer as an additional, new technology for NIH-sponsored research in Wistar's Flow Cytometry Facility. The ImageStream system combines the visual power of microscopy, the quantitative ability of image analysis, and the statistical rigor of flow cytometry in a single platform to create new experimental capabilities. In addition this technology would provide an adjunct tool to have when traditional flow cytometry and microscopy results are either unclear or unexpected. The advantages of the ImageStream as compared to traditional flow cytometry are associated with morphology, providing much of the fluorescent information of a flow cytometer (though at a lower cell throughput rate than flow), but at the same time identifying the location of the signal, which enables the study or tracking of signaling events or the movement of events that happen either on, within or between cells. Also, the ability to associate a spot with its image or images gives the ability to be sure that what you think is a rare event is truly a cell and not a piece of debris. The advantages of the ImageStream as compared to traditional fluorescence microscopy include the ability to look at non-adherent cells in suspension, important because forcing these cells to stick to slides and immobilize them could have strong negative effects on the cells. In addition, the ability to acquire brightfield, darkfield and 4 fluorescent channel images simultaneously on tens to hundreds of cells per second allows for true statistically valid data analysis. Plus, one can quantify signal on thousands of cells while still obtaining individual cell data. Normal microscopy techniques look at only a few hundred cells in a population, making rare event analysis difficult and impractical, if not impossible to do. NIH-funded investigators propose to apply this technology towards: Nuclear Translocation - Quantitative per-cell measurement of nuclear translocation of signaling molecules on large samples;Internalization - Measurement of apoptosis by nuclear morphology change and expression of apoptotic markers;Molecular Co-localization - Quantitative per- cell measurement of the co-association of one or more molecules within or on a cell;Immune Synapse - Quantitative identification of conjugates with molecules recruited to the point of contact between two interacting cells, typically a T cell and an antigen presenting cell (APC). Taken together, the proposed instrumentation usage as justified in this proposal will advance NIH funded research in cancer biology, infectious disease, autoimmunity and vaccine development. PUBLIC HEALTH RELEVANCE: The requested Amnis ImageStream Analyzer instrument will enable Wistar to extend access of state-of-the-art cell analysis in support of multiple NIH-funded projects focused on cellular analysis of transcription, autoimmunity, infectious disease, cancer biology, gene therapy, and vaccine development.